PROJECT SUMMARY/ABSTRACT PIK3CA is the most frequently mutated oncogene in human cancer. PI3K? inhibitors are a new standard of care in PIK3CA mutant ER+ metastatic breast cancer (ER+ MBC) and some patients have durable responses. In this proposal, I capitalize on our recent discovery of a novel mechanism of oncogene activation by double PIK3CA mutations. We have demonstrated that double PIK3CA mutations are frequent across all PIK3CA mutant cancers, occur at recurrent amino acid positions, and are in cis on the same allele. Double mutations activate PI3K signaling and growth more than single hotspot mutations in vitro and in vivo, and biochemically activate PI3K through disruption of p85 inhibition and increased membrane binding. Double mutations increase sensitivity to PI3K? inhibition in cells and in PIK3CA mutant ER+ MBC patients. Our work has uncovered a hierarchical model for the activation and inhibition of PI3K based on mutation number (Vasan, et al. Science 2019). I will test the hypotheses that this hierarchical model extends to PI3K regulation, crosstalk with the estrogen receptor, and activation of novel AKT substrates. I will use recombinant double mutant PI3K complexes reconstituted with Ras and RTK to dissect the mechanisms of regulation (Aim 1.1) and the mechanisms of cellular generation of PIP3 (Aim 1.2). I will leverage knockin and overexpression cellular models to measure activation and inhibition of double mutant ER+ breast cancer (Aim 2.1) and modulation of ER-dependent transcription (Aim 2.2). These experiments will be performed in vitro, in vivo, in patient samples, and under PI3K inhibitor treatment. I will utilize unbiased phosphoproteomics on double mutant cells to credential new AKT protein kinase substrates (Aim 3.1) including the KMT family of histone lysine methyltransferases (Aim 3.2). Together these aims will validate our hierarchical model of oncogene activation by double PIK3CA mutants across a wide variety of cellular and biological processes and will lead to new strategies to inhibit PI3K including testing PI3K? inhibitors in multiple PIK3CA mutant patients. I am an Assistant Attending with the Breast Medicine Service at Memorial Sloan Kettering Cancer Center (MSKCC), and I have outlined a 5-year career plan that builds upon my background studying structural biology and my clinical training in breast oncology. I have assembled an outstanding mentoring team of Dr. Lewis Cantley (primary mentor) and Dr. Maurizio Scaltriti (co-mentor). My advisory committee will include Dr. Neal Rosen, Dr. Ross Levine, and Dr. Komal Jhaveri. They are internationally renowned scientists in their respective fields who will provide me the mentorship and support to attain scientific independence. I will have access to unparalleled institutional support at MSKCC and Weill Cornell Medical College. Both institutions are at the leading edge of cancer research and are heavily invested in career development. Collectively, this training environment will enable me to achieve my goals of ultimately securing a tenure-track independent position.